Role of tumour necrosis factor in the induction of nitric oxide synthase in a rat model of endotoxin shock.

1. This study investigates the role of tumour necrosis factor (TNF) in the induction of nitric oxide synthase (NOS) by bacterial endotoxin (lipopolysaccharide; LPS) in a rat model of endotoxin shock. 2. In anaesthetized rats, pretreatment with a monoclonal antibody for TNF (TNFab; 20 mg kg-1, s.c., at 16 h prior to LPS) ameliorated the fall in mean arterial blood pressure (MAP) in response to LPS (2 mg kg-1, i.v.). For instance, endotoxaemia for 180 min resulted in a fall in MAP from 114 +/- 6 (control) to 84 +/- 5 mmHg (P < 0.01; n = 7). In contrast, animals pretreated with TNFab prior to LPS injection maintained significantly higher MAP when compared to LPS-control (MAP at 180 min; 118 +/- 3 mmHg; P < 0.01, n = 5). 3. Three hours of endotoxaemia was also associated with a significant reduction of the contractile effects of noradrenaline (NA) (10(-8)-10(-6) M) on the thoracic aorta ex vivo. This hyporeactivity to NA was partially restored by in vitro treatment of the vessels with NG-nitro-L-arginine methyl ester (L-NAME, 20 min, 3 x 10(-4) M). Pretreatment of rats with TNFab (20 mg kg-1; at 16 h prior to LPS) significantly (P < 0.05) attenuated the LPS-induced hyporeactivity of rat aortic rings ex vivo. L-NAME did not enhance the contractions of aortic rings obtained from TNFab pretreated LPS-rats. 4. At 180 min after LPS there was a significant elevation of the induced NOS activity in the lung (5.14 +/- 0.57 pmol citrulline mg-1 min-1, n = 8).(ABSTRACT TRUNCATED AT 250 WORDS)     

Aminoguanidine attenuates the delayed circulatory failure and improves survival in rodent models of endotoxic shock.

1. We have investigated the effects of aminoguanidine, a relatively selective inhibitor of the cytokine-inducible isoform of nitric oxide synthase (iNOS), on the delayed circulatory failure, vascular hyporeactivity to vasoconstrictor agents, and iNOS activity in a rat model of circulatory shock induced by bacterial endotoxin (E. coli lipopolysaccharide; LPS). In addition, we have evaluated the effect of aminoguanidine on the 24 h survival rate in a murine model of endotoxaemia. 2. Male Wistar rats were anaesthetized and instrumented for the measurement of mean arterial blood pressure (MAP) and heart rate (HR). Injection of LPS (10 mg kg-1, i.v.) resulted in a fall in MAP from 115 +/- 4 mmHg (time 0, control) to 79 +/- 9 mmHg at 180 min (P < 0.05, n = 10). The pressor effect of noradrenaline (NA, 1 microgram kg-1, i.v.) was also significantly reduced at 60, 120 and 180 min after LPS injection. In contrast, animals pretreated with aminoguanidine (15 mg kg-1, i.v., 20 min prior to LPS injection) maintained a significantly higher MAP (at 180 min, 102 +/- 3 mmHg, n = 10, P < 0.05) when compared to rats given only LPS (LPS-rats). Cumulative administration of aminoguanidine (15 mg kg-1 and 45 mg kg-1) given 180 min after LPS caused a dose-related increase in MAP and reversed the hypotension. Aminoguanidine also significantly alleviated the reduction of the pressor response to NA: indeed, at 180 min, the pressor response returned to normal in aminoguanidine pretreated LPS-rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

The multiple organ dysfunction syndrome caused by endotoxin in the rat: attenuation of liver dysfunction by inhibitors of nitric oxide synthase.

1. We have investigated whether (i) endotoxaemia caused by E. coli lipopolysaccharide in the anaesthetized rat causes a multiple organ dysfunction syndrome (MODS; e.g. circulatory failure, renal failure, liver failure), and (ii) an enhanced formation of nitric oxide (NO) due to induction of inducible NO synthase (iNOS) contributes to the MODS. In addition, this study elucidates the beneficial and adverse effects of aminoethyl-isothiourea (AE-ITU), a relatively selective inhibitor of iNOS activity, and NG-methyl-L-arginine (L-NMMA), a non-selective inhibitor of NOS activity on the MODS caused by endotoxaemia. 2. In the anaesthetized rat, LPS caused a fall in mean arterial blood pressure (MAP) from 117 +/- 3 mmHg (time 0) to 97 +/- 4 mmHg at 2 h (P < 0.05, n = 15) and 84 +/- 4 mmHg at 6 h (P < 0.05, n = 15). The pressor effect of noradrenaline (NA, 1 micrograms kg-1, i.v.) was also significantly reduced at 1 to 6 h after LPS (vascular hyporeactivity). Treatment of LPS-rats with AE-ITU (1 mg kg-1, i.v. plus 1 mg kg-1 h-1 starting at 2 h after LPS) caused only a transient rise in MAP, but significantly attenuated the delayed vascular hyporeactivity seen in LPS-rats. Infusion of L-NMMA (3 mg kg-1, i.v. plus 3 mg kg-1 h-1) caused a rapid and sustained rise in MAP and attenuated the delayed vascular hyporeactivity to NA. Neither AE-ITU nor L-NMMA had any effect on either MAP or the pressor effect elicited by NA in rats infused with saline rather than LPS. 3. Endotoxaemia for 6 h was associated with a significant rise in the serum levels of aspartate or alanine aminotransferase (i.e. GOT or GPT), gamma-glutamyl-transferase (gamma GT), and bilirubin, and hence, liver dysfunction. Treatment of LPS-rats with AE-ITU significantly attenuated this liver dysfunction (rise in GOT, GPT, gamma GT and bilirubin) (P < 0.05, n = 10). In contrast, L-NMMA reduced the increase in the serum levels of gamma GT and bilirubin, but not in GOT and GPT (n = 5). Injection of LPS also caused a time-dependent, but rapid (almost maximal at 2 h), increase in the serum levels of urea and creatinine, and hence, renal dysfunction. This renal dysfunction was not affected by either AE-ITU (n = 10) or L-NMMA (n = 5). In rats infused with saline rather than LPS, neither AE-ITU (n = 4) nor L-NMMA (n = 4) had any significant effect on the serum levels of GOT, GPT, gamma GT, bilirubin, creatinine or urea. 4. Endotoxaemia for 6 h resulted in a 4.5 fold rise in the serum levels of nitrite (9.13 +/- 0.77 microM, P < 0.01, n = 15), which was significantly reduced by treatment with AE-ITU (6.32 +/- 0.48 microM, P < 0.05, n = 10) or L-NMMA (5.10 +/- 0.40 microM, P < 0.05, n = 5). In addition, endotoxaemia for 6 h was also associated with a significant increase in iNOS activity in lung and liver homogenates, which was significantly reduced in lung or liver homogenates obtained from LPS-rats treated with either AE-ITU or L-NMMA. 5. Thus, AE-ITU or L-NMMA (i) inhibits iNOS activity in LPS-rats without causing a significant increase in MAP in rats infused with saline and, hence inhibition of endothelial NOS activity, and (ii) attenuates the delayed circulatory failure as well as the liver dysfunction caused by endotoxaemia in the rat. Thus, an enhanced formation of NO may contribute to the development of liver failure in endotoxic shock.     

Evodia rutaecarpa protects against circulation failure and organ dysfunction in endotoxaemic rats through modulating nitric oxide release

Using a rat model of septic shock we studied the effects of Evodia rutaecarpa, a Chinese herbal medicine with antimicrobial and anti-inflammatory activity, on haemodynamic parameters, biochemical markers of organ function and nitric oxide (NO) production. Anaesthetized rats challenged with a high dosage of endotoxin (Escherichia coli lipopolysaccharide; LPS; 50 mg kg(-1), i.v.) for 6 h showed a severe decrease in mean arterial pressure. This was accompanied by delayed bradycardia, vascular hyporeactivity to phenylephrine and increase in plasma levels of lactate dehydrogenase, aspartate aminotransferase, bilirubin and creatinine, as well as NOx (NO2- plus NO3-). Pretreatment with ethanol extract of E. rutaecarpa (25, 50 and 100 mg kg(-1), i.v.), 1 h before LPS, dose-dependently prevented the circulation failure, vascular hyporeactivity to phenylephrine, prevented liver dysfunction and reduced the NOx over-production in plasma in endotoxaemic rats. A selective inducible NO-synthase (iNOS) inhibitor, aminoguanidine (15 mg kg(-1), i.v.), also effectively ameliorated the above pathophysiological phenomenon associated with endotoxaemia so that the normal condition was approached. Endotoxaemia for 6 h resulted in a significant increase in iNOS activity in the liver homogenate, which was attenuated significantly by E. rutaecarpa pretreatment. In summary, E. rutaecarpa, at the dosages used, exerted these beneficial effects probably through inhibition of iNOS activity and subsequent modulation of the release of NO. These significant results may offer E. rutaecarpa as a candidate for the treatment of this model of endotoxaemia.     

Role of nitric oxide and K+-channels in vascular hyporeactivity induced by endotoxin

This study was to investigate possible mechanisms associated with vascular hyporeactivity to vasoconstrictor agents in rats with endotoxaemia. Wistar-Kyoto rats were anaesthetised and injected with endotoxin [E. coli lipopolysaccharide (LPS); 10 mg/kg, i.v.] for 4 h. Pressor responses to noradrenaline (NA; 1 μg/kg, i.v.) were determined prior to and at every hour after LPS injection. After the in vivo experiment, rat thoracic aortas were excised and prepared as rings 3–4 mm in width. The endothelium was mechanically removed to evaluate K⁺-channel activity and the effects of nitric oxide (NO) on the vascular smooth muscle. Our results demonstrated that: (1) injection of LPS caused a significant fall in blood pressure and a severe vascular hyporeactivity to NA in the anaesthetised rat, (2) the relaxation induced by the K⁺-channel opener cromakalim was greater in rings obtained from endotoxaemic rats and this enhanced relaxation was partially inhibited by pretreatment of these rings with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of the NO/cGMP pathway, (3) endotoxaemia for 4 h was also associated with a profound vascular hyporeactivity to NA ex vivo and this vascular hyporesponsiveness was partially inhibited by ODQ, tetraethylammonium (TEA, a non-selective inhibitor of K⁺-channels) and charybdotoxin [CTX, a selective inhibitor of large conductance calcium-activated K⁺ channels (BK_Ca)], but not by apamin, and (4) the combination of TEA or CTX with ODQ completely restored that vascular responsiveness to normal. These results suggest that activation of BK_Ca and overproduction of NO in the vascular smooth muscle simultaneously contribute to vascular hyporeactivity to vasoconstrictor agents in endotoxaemia. Received: 14 December 1998 / Accepted: 22 March 1999     

Beneficial effect of HCL-31D in murine models of endotoxaemia

We evaluated the effect of HCL-31D, a novel cAMP-specific phosphodiesterase inhibitor, on the induction of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma)-treated rat aortic smooth muscle cells (RASMC) and on survival in a murine model of severe endotoxaemia. Treatment of cultured RASMC with LPS and IFN-gamma resulted in an increase of nitrite, tumour necrosis factor (TNF-alpha) production and induction of iNOS mRNA. However, incubation with HCL-31D (1 approximately 50 microM) for 24 h caused significant attenuation of nitrite and TNF-alpha formation as well as iNOS mRNA induction in a dose-dependent manner but no effect on iNOS activity in RASMC. In addition, administration of HCL-31D (5 mg/kg, i.p.) resulted in that the increase of both plasma nitrate and TNF-alpha levels induced by LPS in vivo was significantly reduced in LPS-treated rats. Treatment of conscious mice with a high dose of LPS (60 mg/kg, i.p.) to ICR mice resulted in a 24-h survival rate of only 10%. However, administration of HCL-31D (5 mg/kg, i.p. at 0 h and 6 h after LPS) improved the 24-h survival to 50%, indicating that HCL-31D has a beneficial effect in murine model endotoxaemia. These effects may be mainly due to inhibition of TNF-alpha formation and of the induction of iNOS. We proposed that the elevation of cAMP levels by HCL-31D may be involved in the prevention of TNF-alpha formation and iNOS induction.     

Attenuation of endotoxin-induced multiple organ dysfunction by 1-amino-2-hydroxy-guanidine, a potent inhibitor of inducible nitric oxide synthase.

1. We have investigated the effects of (i) several guanidines on the activity of the inducible isoform of nitric oxide (NO) synthase (iNOS) in murine cultured macrophages and rat aortic vascular smooth muscle cells (RASM); and (ii) 1-amino-2-hydroxy-guanidine, the most potent inhibitor of iNOS activity discovered, on haemodynamics, multiple organ (liver, renal, and pancreas) dysfunction and iNOS activity in rats with endotoxic shock. 2. The synthesized guanidine analogues caused concentration-dependent inhibitions of the increase in nitrite formation caused by lipopolysaccaride (LPS, 1 microgram ml-1) in J774.2 macrophages and RASM cells with the following rank order of potency: 1-amino-2-hydroxy-guanidine > 1-amino-2-methyl-guanidine > 1-amino-1-methyl-guanidine > 1-amino-1,2-dimethyl-guanidine. Interestingly, 1-amino-2-hydroxy-guanidine (IC50: J774.2, 68 microM; RASM, 114 microM) was more potent in inhibiting nitrite formation caused by LPS than NG-methyl-L-arginine, but less potent than aminoethyl-isothiourea. 3. In the anaesthetized rat, LPS caused a fall in mean arterial blood pressure (MAP) from 115 +/- 4 mmHg (time 0) to 98 +/- 5 mmHg at 2 h (P < 0.05, n = 10) and 69 +/- 5 mmHg at 6 h (P < 0.05, n = 10). The pressor effect of noradrenaline (NA, 1 mg kg-1, i.v.) was also significantly reduced at 1 to 6 h after LPS (vascular hyporeactivity). Treatment of LPS-rats with 1-amino-2-hydroxy-guanidine (10 mg kg-1, i.v. plus 10 mg kg-1 h-1 starting at 2 h after LPS) prevented the delayed hypotension and vascular hyporeactivity seen in LPS-rats. However, 1-amino-2-hydroxy-guanidine had no effect on either MAP or the pressor effect elicited by NA in rats infused with saline rather than LPS. 4. Endotoxaemia for 6 h caused a significant rise in the serum levels of aspartate or alanine aminotransferase (i.e. GOT or GPT) and bilirubin, and hence, liver dysfunction. Treatment of LPS-rats with 1-amino-2-hydroxy-guanidine significantly attenuated the liver dysfunction caused by LPS (P < 0.05, n = 10). Injection of LPS also caused a rapid (almost maximal at 2 h) increase in the serum levels of urea and creatinine, and hence, renal dysfunction. This renal dysfunction was not affected by 1-amino-2-hydroxy-guanidine (P > 0.05; n = 10). Endotoxaemia also caused a dysfunction of pancreas (rise in serum levels of lipase) as well as a metabolic acidosis (falls in PCO2, HCO3 and base excess). Both pancreatic dysfunction and metabolic acidosis were largely attenuated by treatment of LPS-rats with 1-amino-2-hydroxy-guanidine. In rats infused with saline rather than LPS, 1-amino-2-hydroxy-guanidine had no effect on liver, renal or pancreatic function (n = 4). 5. Endotoxaemia for 6 h resulted in a rise in the serum levels of nitrite (11.0 +/- 0.8 microM, P < 0.01, n = 10), which was significantly reduced by 1-amino-2-hydroxy-guanidine (6.5 +/- 0.7 microM, P < 0.05, n = 10). Endotoxaemia for 6 h was also associated with a significant increase in iNOS activity in lung and liver, which was significantly reduced in lung or liver homogenates obtained from LPS-rats treated with 1-amino-2-hydroxy-guanidine. In addition, endotoxaemia for 6 h resulted in a significant increase in myeloperoxidase activity (MPO), an indicator of neutrophil infiltration, in the liver. Treatment of LPS-rats with 1-amino-2-hydroxy-guanidine did not affect the rise in MPO-activity in the liver caused by endotoxin. 6. Thus, 1-amino-2-hydroxy-guanidine is a potent inhibitor of iNOS activity in macrophages or RASM in culture as well as in rats with endotoxic shock. Inhibition of iNOS activity with 1-amino-2-hydroxy-guanidine prevents the delayed circulatory failure and attenuates the dysfunction of liver, and pancreas, as well as the metabolic acidosis caused by endotoxaemia.     

Effect of selective blockade of endothelin ETB receptors on the liver dysfunction and injury caused by endotoxaemia in the rat.

1. We investigated the effects of the selective endothelin (ET)A receptor antagonist BQ-485 and the selective ETB receptor antagonist BQ-788 on circulatory failure, multiple organ dysfunction syndrome (MODS) and the alterations in acid base balance caused by endotoxaemia in the anaesthetized rat. 2. Male Wistar rats were anaesthetized (thiopentone sodium; 120 mg kg-1, i.p.) and received a continuous infusion of vehicle (saline, 0.6 ml kg-1h-1, i.v.), BQ-485 (10 nmol kg-1 min-1, i.v.) or BQ-788 (10 nmol kg-1 min-1, i.v.). Fifteen min later, animals received a bolus injection of either saline (0.9% NaCl, 1 ml kg-1, i.v.) or E. coli lipopolysaccharide (LPS, 10 mg kg-1, i.v.). 3. Injection of LPS resulted in a fall in blood pressure from 115 +/- 4 mmHg (time 0) to 82 +/- 4 mmHg at 360 min (n = 15) as well as a hyporeactivity to the pressor responses to noradrenaline (NA, 1 microgram kg-1, i.v.). Infusion of BQ-788 attenuated the delayed hypotension (at 360 min: 100 +/- 4 mmHg, n = 7; P < 0.05) and significantly enhanced the pressor responses elicited by NA (at 60 to 240 min). In contrast, treatment of LPS-rats with BQ-485 augmented the hypotension (at 360 min), but did not affect the vascular hyporeactivity elicited by endotoxaemia. 4. Endotoxaemia for 360 min resulted in rises in the serum levels of urea and creatinine (indicators of renal failure), glutamate-oxalate-transferase (GOT) and glutamate-pyruvate-transferase (GPT) (indicators of hepatocellular injury), and bilirubin and gamma-glutamyl transferase (gamma GT) (indicators of liver failure) as well as nitrite (indicator of the induction of nitric oxide synthase; iNOS). Treatment of LPS-rats with BQ-788, but not with BQ-485, attenuated the degree of liver injury and failure, while neither BQ-788 nor BQ-485 affected the acute renal failure or the induction of iNOS caused by endotoxin. 5. Endotoxaemia also caused (within 15 min) an acute metabolic acidosis (falls in pH, HCO3-and base excess) which was compensated by hyperventilation (fall in PaCO2). Treatment of LPS-rats with BQ-788 or BQ-485 did not affect the metabolic acidosis caused by LPS. 6. Thus, the selective ETB receptor antagonist BQ-788 attenuated (i) the delayed hypotension, (ii) the vascular hyporeactivity to NA as well as (iii) the degree of hepatocellular injury and dysfunction caused by endotoxin in the anaesthetized rat. In contrast, the selective ETA receptor antagonist did neither attenuate the circulatory failure nor the liver or renal dysfunction associated with endotoxaemia. We propose that the prevention of the hepatocellular dysfunction and injury caused BQ-788 in endotoxaemia is due to an improvement in oxygen delivery to the liver secondary to (i) inhibition of pre-sinusoidal constriction, (ii) inhibition of sinusoidal constriction, and (iii) improvement in perfusion pressure.     

Effects of the endothelin receptor antagonist, SB 209670, on circulatory failure and organ injury in endotoxic shock in the anaesthetized rat.

1. This study investigates the effects of the non-selective ETA/ETB receptor antagonist, SB 209670, on systemic haemodynamics, renal function, liver function, acid-base balance and survival in a rat model of endotoxic shock. 2. Injection of E. coli lipopolysaccharide (LPS, 10 mg kg-1, i.v.) resulted in increases in the serum levels of tumour necrosis factor-alpha (TNF-alpha, maximum 60 min after LPS), endothelin-1, (ET-1; maximum 120 min after LPS), and interferon-gamma (IFN-gamma, maximum 180 min after LPS). 3. Injection of LPS also resulted in a fall in blood pressure from 113 +/- 3 mmHg (time = 0) to 84 +/- 4 mmHg at 360 min (n = 15) as well as a hyporeactivity to the vasoconstrictor responses elicited by noradrenaline (NA, 1 microgram kg-1, i.v.). Pretreatment of rats with a continuous infusion of SB 209670 (3 mg kg-1, i.v. bolus + 100 micrograms kg-1, i.v. infusion commencing 15 min prior to LPS) significantly augmented the hypotension as well as the vascular hyporeactivity to NA caused by endotoxaemia. 4. Pretreatment of LPS-rats with SB 209670 (3 mg kg-1, i.v. bolus given 15 min prior to LPS) or infusion of SB 209670 (bolus dose and infusion as above) resulted in a reduction in 6 h-survival from 71% (control) to 30% and 13%, respectively. 5. Endotoxaemia for 4 h resulted in rises in the serum levels of urea and creatinine (indicators of renal failure), but not in the serum levels of bilirubin, GPT and GOT (indicators of liver dysfunction and/or hepatocellular injury). Pretreatment of LPS-rats with SB 209670 (3 mg kg-1, i.v. bolus 15 min prior to LPS) significantly augmented the serum levels of creatinine, bilirubin, GPT and GOT caused by endotoxin. In addition, endotoxaemia caused, within 15 min, an acute metabolic acidosis (falls in pH, HCO3- and base excess) which was compensated by hyperventilation (fall in PaCO2). Pretreatment of LPS-rats with SB 209670 (3 mg kg-1, i.v. bolus) significantly augmented the metabolic acidosis caused by LPS. 6. Thus, the non-selective ETA/ETB receptor antagonist, SB 209670, augments the degree of (i) hypotension, (ii) vascular hyporeactivity to noradrenaline, (iii) renal dysfunction and (iv) metabolic acidosis caused by endotoxin in the anaesthetized rat. In contrast to rats treated with LPS alone, LPS-rats treated with SB 209670 exhibited liver dysfunction and hepatocellular injury. We propose that the release of endogenous ET-1 serves to maintain blood pressure and subsequently organ perfusion in septic shock.     

Recombinant human erythropoietin inhibits iNOS activity and reverts vascular dysfunction in splanchnic artery occlusion shock.

1. We investigated the effects of recombinant human erythropoietin (rh-EPO) in splanchnic artery occlusion (SAO) shock. Sham operated animals were used as controls. Survival rate, mean arterial blood pressure (MAP), serum Tumor Necrosis Factor (TNF-alpha), plasma nitrite/nitrate concentrations, red blood cell (RBC) count, blood haemoglobin (Hb), the responsiveness of aortic rings to phenylephrine (PE, 1 nM-10 microM) and the activity of inducible nitric oxide synthase (iNOS) were studied. 2. SAO shocked rats had a decreased survival rate (0% at 4 h of reperfusion, while sham shocked rats survived more than 4 h), enhanced serum TNF-alpha concentrations, increased plasma nitrite/nitrate levels (60+/-9.5 microM; sham shocked rats= 2+/-0.4 microM), decreased MAP, unchanged RBC count and blood Hb and enhanced iNOS activity in the aorta. Moreover aortic rings from shocked rats showed a marked hyporeactivity to PE. 3. Rh-EPO (25, 50 and 100 U 100 g(-1), 5 min following the onset of reperfusion) increased survival rate (70% at 4 h of reperfusion with the highest dose), reduced plasma nitrite/nitrate concentrations (10.3+/-3.3 microM), increased MAP, did not change RBC count and blood Hb, and inhibited iNOS activity in thoracic aortae. Furthermore rh-EPO, either in vivo or in vitro (10 U for 1 h in the organ bath), restored to control values the hyporeactivity to PE. Finally rh-EPO inhibited the activity of iNOS in peritoneal macrophages activated with endotoxin. 4. Our data suggest that rh-EPO protects against SAO shock by inhibiting iNOS activity.     

Cutaneous venous dysfunction studied in vivo in the LPS-treated rabbit: implication of NO in saphenous vein hyporeactivity

Superficial vein pathology involves both mechanical (hyperpressure and distension) and inflammatory mechanisms. Conflicting results exist about the role of NO in the venous hyporeactivity induced by inflammation. In order to clarify this point, we aimed to investigate the effects of sepsis on cutaneous vein responsiveness in vivo and the possible contributions of constitutive and inducible NOS to the changes of venous contractility. Saphenous vein diameter was recorded by an ultrasonic echo-tracking device in pentobarbital-anaesthetised rabbits. Bacterial lipopolysaccharide (LPS) was administered i.v. at 20 mg/kg/15 min, inducing a progressive fall in mean arterial blood pressure after 2-3 h. The effects of LPS on saphenous vein responsiveness to noradrenaline (2 microg/kg i.v.) were measured simultaneously. In some rabbits, veins were removed for immunochemistry to detect iNOS staining. The venoconstriction to noradrenaline was already significantly reduced at 30 min after LPS (6+/-1% instead of 19+/-1% before LPS) and was completely abolished 3 h after LPS. A reduction of the venoconstriction induced by sumatriptan, a 5-HT(1B/D) agonist, (100 microg/kg, 11+/-1% after saline n=5) was also observed 180 min after LPS infusion (3+/-1%, n=4). The venodilatations induced by acetylcholine or sodium nitroprusside injected locally into the vein were not altered by LPS. When administered 90 min after LPS infusion, the NOS inhibitor L-NAME but not the selective iNOS inhibitor L-NIL (10 mg/kg) induced a recovery of the venoconstriction. Preventive perfusion with L-NAME (10 mg/kg/2 h) reduced the initial hyporeactivity to noradrenaline (30 to 60 min), but accelerated the lethal fall in MAP. L-NIL (10 mg/kg/2 h), to a lesser extent than L-NAME, also reduced the initial hyporeactivity to noradrenaline; in contrast to L-NAME, L-NIL also delayed the complete loss of noradrenaline constriction and improved animal survival. In control animals, neither L-NAME nor L-NIL modified the venoconstriction induced by noradrenaline. iNOS staining was observed in the saphenous vein endothelium after LPS. The experimental model developed in these experiments allows the study of venous responsiveness during sepsis in vivo. Our results show that LPS administration reduces saphenous vein contractility to both adrenergic and serotoninergic constrictor agents. The data suggest that both endothelial and inducible NO are involved in the loss of venous reactivity but these enzymes exert contrasting effects on blood pressure changes.     

Cardiac and regional haemodynamics, inducible nitric oxide synthase (NOS) activity, and the effects of NOS inhibitors in conscious, endotoxaemic rats.

1. A reproducible model of the hyperdynamic circulatory sequelae of endotoxaemia in conscious, chronically-instrumented Long Evans rats, was achieved with a continuous infusion of lipopolysaccharide (LPS, 150 micro g kg(-1) h(-1)) for 32 h. Over the first 2 h of LPS infusion, there was a transient hypotension and tachycardia, accompanied by a marked increase in renal flow and vascular conductance, although there were reductions in cardiac and stroke index. Between 4-8 after the start of LPS infusion, there was slight hypotension and tachycardia, and a transient rise in mesenteric flow and conductance, but reductions in the hindquarters vascular bed; the hyperaemic vasodilatation in the renal vascular bed was maintained. At this stage, all cardiac haemodynamic variables were not different from baseline. At this stage, cardiac and stroke index were substantially elevated, in association with marked increases in peak aortic flow, dF/dtmax and total peripheral conductance; these changes were well-maintained over the following 8 h of LPS infusion. 2. By 2 h after the start of LPS infusion, only lung inducible nitric oxide synthase (iNOS) activity was increased, but at 6 h there were significant increases in iNOS activity in lung, liver, spleen, heart and aorta. (43.3 +/- 7.8, 28.8 +/- 3.3, 50.8 +/- 7.2, 3.04 +/- 0.29, 3.76 +/- 0.94 pmol min(-1) mg(-1) protein, respectively). However, by 24 h after the start of LPS infusion, iNOS activity was not elevated significantly in any tissue examined, and kidney iNOS activity did not change significantly during LPS infusion. Plasma nitrite/nitrate levels were increased after 2 h infusion of LPS (from 6.07 +/- 1.23 to 29.44 +/- 7.08 micromol l(-1)), and further by 6 h (228.10 +/- 29.20 micromol l(-1)), but were less 24 h after onset of LPS infusion (74.96 +/- 11.34 micromol l(-1)). Hence, the progressive hypotension, increasing cardiac function and developing hyperaemic vasodilatation in renal and hindquarters vascular beds between 8-24 h after the onset of LPS infusion, occurred when tissue iNOS activity and plasma nitrite/nitrate levels were falling. 3. Pretreatment with NG-monomethyl-L-arginine (L-NMMA, 30 mg kg(-1) bolus, 30 mg kg(-1) h(-1) infusion) 1 h before LPS infusion did not prevent the early hypotension, but abolished the initial renal vasodilatation and the later (6-8 h) fall in mean arterial pressure (MAP), and the additional renal vasodilatation.     

Delayed circulatory failure due to the induction of nitric oxide synthase by lipoteichoic acid from Staphylococcus aureus in anaesthetized rats.

1. This study investigates the effect of lipoteichoic acid (LTA) from the cell wall of Staphylococcus aureus, a micro-organism without endotoxin, on haemodynamics and induction of nitric oxide synthase (iNOS) in the anaesthetized rat. 2. Intravenous injection of LTA (10 mg kg-1) resulted in a decrease in blood pressure from 123 +/- 1 mmHg to 83 +/- 7 mmHg after 270 min (P < 0.001) and a reduction of the pressor response to noradrenaline (1 microgram kg-1) from 33 +/- 1 mmHg.min to 23 +/- 3 mmHg.min after 270 min (P < 0.05). 3. The delayed circulatory failure (hypotension and vascular hyporeactivity) caused by LTA was prevented by pretreatment of rats with dexamethasone (10 mg kg-1, 60 min prior to LTA) or the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 10 mg kg-1 h-1, i.v. infusion starting 30 min prior to LTA). 4. In contrast, treatment of rats with polymyxin B (0.05 mg kg-1), an agent which binds endotoxin (lipopolysaccharides, LPS), did not affect the delayed circulatory failure caused by LTA. Polymyxin B, however, attenuated the hypotension and vascular hyporeactivity to noradrenaline afforded by endotoxaemia (2 mg kg-1 LPS, i.v.) for 270 min. 5. The delayed circulatory failure caused by LTA was associated with a time-dependent increase in (i) the expression of iNOS protein in the lung (Western blot analysis), and (ii) iNOS activity. This increase in iNOS protein and activity was prevented by pretreatment of LTA-rats with dexamethasone (10 mg kg-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Antioxidant eugenosedin-A protects against lipopolysaccharide-induced hypotension, hyperglycaemia and cytokine immunoreactivity in rats and mice

Eugenosedin-A has been demonstrated to possess alpha/beta-adrenoceptor and serotonergic receptor blocking activities. We have investigated by what mechanisms eugenosedin-A prevents lipopolysaccharide (LPS)-induced hypotension, vascular hyporeactivity, hyperglycaemia, oxidative injury or inflammatory cytokines formation in rats. Intravenous administration of eugenosedin-A, trazodone, yohimbine (1 mg kg(-1)), aminoguanidine or ascorbic acid (15 mg kg(-1)) normalized LPS (10 mg kg(-1))-induced hypotension. Pretreatment with eugenosedin-A or the other agents 30 min before LPS injection reduced aortic hyporeactivity. LPS-induced increases in plasma interleukin-1beta (IL-beta), IL-6, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and blood glucose levels were significantly inhibited by eugenosedin-A (1 mg kg(-1), i. v.). The same dose of trazodone, a chloropiperazinylbenzene-type antidepressant, and yohimbine, an alpha(2)-adrenoceptor antagonist, reduced IL-1beta and TNF-alpha, but it could not inhibit hyperglycaemia. Aminoguanidine, an inducible nitric oxide synthase (iNOS) inhibitor, and ascorbic acid, an antioxidant, decreased IL-1beta, TNF-alpha contents and hyperglycaemia. Eugenosedin-A and the other agents inhibited Fe(2+)-ascorbic acid-induced peroxidation in rat cortex, indicating that those agents had antioxidant effects, with the exception of aminoguanidine. In free radical scavenged experiments, eugenosedin-A and ascorbic acid eliminated peroxyl radicals. All test agents inhibited the LPS-induced increase of malondialdehyde (MDA) content in rat brain homogenates. When mice were administered an intraperitoneal injection of LPS alone, mortality occurred from 4 to 16 h, after which time all were dead. However, eugenosedin-A significantly prolonged the survival time after LPS injection, suggesting that eugenosedin-A protected against LPS-induced cardiovascular dysfunction, hyperglycaemia, tissue injury and inflammatory cytokine production. This was attributable mainly to the antioxidant and peroxyl radical scavenged effects of eugenosedin-A, and which may be, at least in part, due to its blockade on alpha/beta-adrenergic and serotonergic receptors.     

Effect of anthocyanins contained in a blackberry extract on the circulatory failure and multiple organ dysfunction caused by endotoxin in the rat

Anthocyanins are a group of naturally occurring phenolic compounds related to the colouring of plants, flowers and fruits. These pigments are important as quality indicators, chemotaxonomic markers and for their antioxidant activities. Here we have investigated the therapeutic efficacy of anthocyanins contained in a blackberry extract on (i) circulatory failure, (ii), multiple organ dysfunction and (iii) activity of the inducible isoforms of nitric oxide (NO) synthase (iNOS) and cyclooxygenase (COX-2) in anaesthetised rats with endotoxic shock. In a model of endotoxic shock induced by lipopolysaccharide (LPS, E. coli, 10 mg/kg, i.v.) in the rat, pretreatment with anthocyanins present in the blackberry extract (5 mg/kg, i. v. 30 min before LPS) prevented the hypotension induced by LPS. Endotoxaemia also caused rises in the serum levels of (i) glutamyl oxaloacetic transaminase (GOT), glutamyl pyruvic transaminase (GPT), alkaline phosphates and bilirubin (hepatic dysfunction) (ii) creatinine (renal dysfunction), (iii) amylase and lipase (pancreatic injury), (iii) NOx and 6-keto-PGF1 alpha. Anthocyanins attenuated the hepatic and pancreatic injury, the renal dysfunction and decreased NOx and 6-keto-PGF1 alpha levels. Endotoxaemia for 6 h resulted in a substantial increase in iNOS and COX activity in rat lung, which was attenuated in rats pretreated with anthocyanins. Moreover, anthocyanins (0.02 - 0.32 mg/mL) inhibited in vitro iNOS and COX activity from lung of LPS-treated rats. Polymorphonuclear (PMN) infiltration (myeloperoxidase activity), lipid peroxidation (malondialdehyde levels), as well as tissue injury (histological examination) induced by LPS in rat lung and ileum was reduced by anthocyanins (5 mg/kg, i. v. 30 min before LPS). Furthermore, endotoxaemia induced the formation of nitrotyrosine and poly(ADP-ribose) synthetase (PARS) activation as determined by immunohistochemical analysis of lung and ileum tissues. The degree of staining was lowered by anthocyanin treatment. These results indicate that the anthocyanins contained in the blackberry extract exert multiple protective effects in endotoxic shock.     

Eugenosedin-A amelioration of lipopolysaccharide-induced up-regulation of p38 MAPK, inducible nitric oxide synthase and cyclooxygenase-2

In this study, we investigate the protective effects of eugenosedin-A on p38 mitogen-activated protein kinase (MAPK), inflammatory nitric oxide (NO) and cyclooxygenase-2 (COX-2) pathways in a rat model of endotoxin shock. Rats were pretreated with eugenosedin-A, trazodone, yohimbine (1 mg kg(-1), i.v.), aminoguanidine or ascorbic acid (15 mg kg(-1), i.v.) 30 min before endotoxin challenge. Endotoxaemia was induced by a single i.v. injection of lipopolysaccharide (LPS, 10 mg kg(-1)). In rats not treated with eugenosedin-A, LPS increased plasma concentrations of NO and prostaglandin E(2) (PGE(2)), and levels of p38 MAPK, inducible NO synthase (iNOS) and COX-2 proteins in the liver, lung, aorta and lymphocytes. In the pre-treated rats, eugenosedin-A not only inhibited the LPS-induced NO and PGE(2) levels but also attenuated the LPS-induced increase in p38 MAPK and iNOS levels in the liver, aorta and lymphocytes. Eugenosedin-A also reduced LPS-induced COX-2 proteins in the aorta and lymphocytes. Likewise, aminoguanidine, ascorbic acid, yohimbine and trazodone were also found to decrease NO and PGE(2) concentrations after endotoxin challenge. While aminoguanidine and ascorbic acid also attenuated the LPS-induced increase in p38 MAPK, iNOS and COX-2 proteins in the aorta and lymphocytes, trazodone and yohimbine inhibited only the increase in p38 MAPK, iNOS and COX-2 proteins in lymphocytes. Finally, eugenosedin-A (10(-10)-10(-8) M) significantly inhibited the biphasic response induced by hydrogen peroxide (10(-6)-3 x 10(-5) M) in rat denudated aorta. Taken together, the results of this study indicate that eugenosedin-A, as well as ascorbic acid, can attenuate free-radical-mediated aortic contraction and relaxation. It may therefore be able to reduce the damage caused by septic shock by inhibiting formation of p38 MAPK, iNOS, COX-2 and free radicals.     

Pressor and vasoconstrictor effects of methylene blue in endotoxaemic rats

Nitric oxide (NO) is a primary mediator of hypotension in sepsis. We examined the effects of methylene blue (MB), an inhibitor of the NO/cGMP pathway, on mean arterial pressure (MAP), cardiac output (CO), total peripheral resistance (TPR), mesenteric blood flow (MBF) and renal blood flow (RBF) in pentobarbitone-anaesthetised rats injected with lipopolysaccharide (LPS, 7.5 mg/kg). MB (1, 3 or 10 mg/kg.h) or vehicle was i.v. infused into four groups at 2.5 h after i.v. injection of LPS. Two other groups received MB or vehicle at 2.5 h after receiving saline. LPS reduced MAP, CO, RBF as well as MBF at 2.5 and 4 h, and increased TPR at 2.5 but not 4 h. Whereas MB alone had no effects on measured variables in control rats at 4 h, in LPS-treated rats, it elevated TPR at all doses and attenuated the fall in MAP at the two low doses. CO was unaltered by low doses of MB but reduced by the high dose. MBF was unaltered, but RBF was increased by the lowest dose but decreased by the highest dose of MB. Therefore, in endotoxaemia, a low dose of MB increases MAP and TPR but does not alter CO; a high dose of MB does not raise MAP but increases TPR and reduces CO. Received: 13 December 1997 / Accepted: 23 February 1998     

The lazaroid, U-74389G, inhibits inducible nitric oxide synthase activity, reverses vascular failure and protects against endotoxin shock

The aim of our study was to investigate the effect of the 21-aminosteroid U-74389G [21- < 4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl-pregna-1,4,9,(11) triene-3,20-dione(z)-2-butenedionate] on the l-arginine-nitric oxide (NO) pathway in a rat model of endotoxin shock. Endotoxin shock was produced in male rats by a single intravenous (i.v.) injection of 20 mg/kg of Salmonella Enteritidis lipopolysaccharide (LPS). Rats were treated with U-74389G (7.5, 15 and 30 mg/kg i.v.) or vehicle (1 ml/kg i.v.) 5 min after endotoxin challenge. Lipopolysaccharide administration reduced survival rate (0%, 72 h after endotoxin administration) decreased mean arterial blood pressure, enhanced plasma concentration of bilirubin and alanine aminotransferase and increased plasma nitrite concentrations. Lipopolysaccharide injection also increased the activity of inducible NO synthase in the liver and in the aorta. Furthermore aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (1 nM-10 microM). In addition lipopolysaccharide (50 microg/ml for 4 h) in vitro stimulation significantly increased nitrite production in peritoneal macrophages harvested from normal rats. Treatment with U-74389G (15 and 30 mg/kg i.v., 5 min after endotoxin challenge) significantly protected against lipopolysaccharide-induced lethality (90% survival rate 24 h and 80% 72 h after lipopolysaccharide injection, respectively, following the highest dose of the drug), reduced hypotension, ameliorated liver function, decreased plasma nitrite levels, restored the hyporeactivity of aortic rings to their control values and inhibited the activity of inducible NO synthase in the liver and in the aorta. Finally, U-74389G in vitro (12.5, 25 and 50 microM) significantly inhibited nitrite production in endotoxin stimulated peritoneal macrophages. The data suggest that U-74389G may exert beneficial effects in an experimental model of septic shock by inhibiting the activity of the inducible NO synthase.     

Inhibition of extracellular signal-regulated kinase (ERK1/2) activity reverses endotoxin-induced hypotension via decreased nitric oxide production in rats.

Overproduction of reactive oxygen and nitrogen species leads to oxidative stress and decreased total antioxidant capacity, which is responsible for high mortality from several inflammatory diseases such as endotoxic shock. Among reactive nitrogen species, nitric oxide (NO) produced by inducible NO synthase (iNOS) during endotoxemia is the major cause of vascular hyporeactivity, hypotension and multiple organ failure. This study was conducted to determine if mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK1/2) contributes to endotoxin-induced hypotension as well as vascular inflammation and oxidative stress via NO production. In conscious male Wistar rats, injection of endotoxin (10 mg kg(-1), i.p.) caused a decrease in mean arterial pressure (MAP) for 4h and increased levels of nitrite in serum, aorta and mesenteric artery. These effects of endotoxin were prevented by selective inhibition of ERK1/2 phosphorylation by MAPK kinase (MEK1/2) with U0126 (5 mg kg(-1), i.p.; 1h after endotoxin). Endotoxin also caused an increase in protein levels of phosphorylated ERK1/2 in aorta which was abolished by U0126. Selective inhibition of iNOS with phenylene-1,3-bis[ethane-2-isothiourea] dihydrobromide (1,3-PBIT) (10 mg kg(-1), i.p.; 1h after endotoxin) did not change the endotoxin-induced increase in ERK1/2 activity. Myeloperoxidase activity was increased in aorta and decreased in mesenteric artery by endotoxin, which was reversed by U0126. Endotoxin-induced decrease in one of the products of lipid peroxidation, malonedialdehyde (MDA) was prevented by U0126 in mesenteric artery; however, U0126 caused a further decrease in the levels of MDA in aorta. These data suggest that increased phosphorylation of ERK1/2 by MEK1/2 contributes to the endotoxin-induced hypotension via NO production rat aorta and mesenteric artery. It is likely that ERK1/2 mediates the effect of endotoxin on MPO activity in a different degree in the tissues suggesting possible involvement of any mediator and/or mechanism which also causes neutrophil infiltration during inflammatory response at least in mesenteric artery. Moreover, ERK1/2 seems to be involved in the endotoxin-induced increase in total antioxidant capacity in mesenteric artery.